Project Summary Muscle trauma is one of the most common and repetitive types of injury in humans. Owing to the regenerative functions of Muscle Stem Cells (MuSCs), the injured myotome can fully regain its structural and functional integrity within just a few days. In addition to this remarkable regenerative function, MuSCs also play a significant role in the maintenance of muscle homeostasis under the normal condition. On the other hand, the decline in MuSCs function is implicated in muscle wasting diseases and several forms of muscular dystrophy. The functions of MuSCs are influenced by the niche it occupies. For example, the niche emits signals to regulate MuSCs quiescence in the uninjured state, or activation when regeneration is required. Understanding how these signaling pathways control quiescence and activation of MuSCs is essential to devise the next generation of therapeutic approaches for diseases of MuSCs dysfunction. We recently described a novel signaling pathway in MuSCs that stimulates regenerative myogenesis by epigenetically activating the transcription of the Myog promoter. In this pathway, Per-Arnt-Sim domain Kinase (PASK) phosphorylates WD40-domain repeat 5 (Wdr5) at the onset of the myogenesis program. Phosphorylated Wdr5 stimulates the Myog transcription by inducing histone H3 trimethylation at lysine 4 (H3K4me3) and the recruitment of the MyoD transcription factor at the Myog promoter. We present evidence that PASK expression and activity is induced in regenerating myoblasts by signaling cues such as insulin and nutrients in mTOR complex 1 dependent manner. Functionally, loss of Pask in mice retards regenerative myogenesis, and over-expression of PASK depletes the stem cells. Thus, we hypothesize that PASK plays a role balancing self-renewal vs differentiation during regeneration. Building upon these data, in this proposal, we will investigate how PASK expression and activity is regulated in the regenerating myoblast (Specific Aim 1). In activated MuSCs, PASK phosphorylated Wdr5 induces H3K4me3 modification, MyoD recruitment and nucleosomal remodeling on the Myog promoter yet the mechanism is not clear. In Specific Aim 2, we will extensively study two interacting partners of Wdr5 which could remodel the Myog promoter for its activation. Upon induction of Myogenin, Pax7 expression is down-regulated and terminal differentiation is established. Since PASK is an upstream regulator of the Myog expression, we are interested determining how loss or gain of PASK affects the self-renewing population during regeneration and normal aging process, which we will study in Specific Aim 3. Ultimately, these aims will allow us to understand how niche signaling cues control the balance between self-renewal and differentiation and how it can be exploited for improvement of physiological conditions such as sarcopenia.